Malignant brain tumors are the leading cause of cancer-related death in people under the age of 35. Immunotherapy in the form of personalized vaccines has demonstrated immunologic activity and clinical responses in select glioma patients with minimal toxicity. There are numerous ongoing clinical trials that utilize cultured tumor cells as the source of vaccine antigen for the treatment of a wide array of tumors. However, very little is known about how cell culture conditions affect the immune response and ultimate clinical response following vaccination. The consistent condition used is expansion of tumor cells in atmospheric oxygen (20% O2). We have identified the oxygen concentration used in tissue culture as a primary determinant of the immunogenicity of tumor cell vaccines. Our data have led to the attractive central hypothesis that the oxygen tension in a tumor cell culture acts as a master immunologic switch, dictating the type and strength of immune response induced by vaccination. We have reproducibly shown that lysates from glioma cells cultured in 5% O2 prime cytotoxic T lymphocytes (CTLs) with superior effector functions relative to lysate from glioma cells cultured in 20% O2 in human and murine systems. This difference profoundly affects the efficacy of immunotherapy, as shown by significant improvements in survival in murine models of glioma and breast carcinoma. We demonstrated that administration of 5% O2 lysate vaccination caused superior CTL proliferation, cytokine elaboration, tumoricidal function, and trafficking to tumor sites relative to 20% O2 lysate vaccines. Conversely, 20% O2 lysate vaccines enhance antibody responses. Despite reduced tumor-reactive antibody responses, the 5% O2 vaccines require B cells for therapeutic efficacy, revealing a putative role of B cells in CTL priming. Additionally, we have evidence that glioma cells grown in 5% O2 upregulate toll-like receptor (TLR) 2 ligands because TLR2 is required in several systems to distinguish the immunogenicity of 5% and 20% O2 lysates. The goal of this proposal is to elucidate the molecular basis of the oxygen switch effect in order to rationally improve the efficacy of tumor cell vaccines. In specific aim 1 we will determine and optimize tumor cell intrinsic changes that modulate immunogenicity. Pharmacologic strategies to increase hypoxia inducible factors (HIF) in tumor cells will be compared to actual hypoxia in cell cultures that will subsequently be assayed for immunogenicity. Additionally, we will test the hypothesis that HIF2a is the molecular switch that induces expression of TLR2 ligands. In specific aim 2, we will test our hypothesis that B cells are required because IgG-tumor lysate complexes trigger Fc receptor-mediated cross presentation of antigens to enhance CTL responses. In specific aim 3, we will determine if the adjuvant effect of 5% O2 lysates is due to expression of Annexin A2, a novel putative TLR2 ligand we recently found to be upregulated in hypoxia. Collectively, this knowledge will facilitate groundbreaking approaches to enhance the efficacy of immunotherapy for glioma and other non-central nervous system tumors.